ALBERT

Description: Myelodysplastic syndrome (MDS) and chronic myelomonocytic leukaemia (CMML) are haematological disorders that develop in haematopoietic stem or progenitor cells (HSPCs) and are characterised by ineffective haematopoiesis. 5'-Azacitidine (AZA) is a DNA demethylating agent that is effective in treating MDS and CMML. However, response rates are less than 50% and the basis for poor response is currently unknown. A patient's potential to respond cannot be currently determined until after multiple cycles of AZA treatment and alternative treatment options for poor responders are limited. To address these fundamental questions, we enrolled patients on a compassionate access program prior to the listing of AZA on the pharmaceuticals benefit scheme in Australia. We have collected bone marrow from 18 patients (10 MDS, 8 CMML) at seven different stages of treatment, starting from before treatment until after six cycles of AZA treatment, and isolated high-purity CD34+ HSPCs at each stage. 10 of these patients (5 MDS and 5 CMML) responded completely to AZA while 8 did not achieve complete response. We performed next-generation sequencing (RNA-seq) of these HSPCs to identify the basis of poor response to AZA therapy. Analysis of the RNA-seq data from pre-treatment HSPCs has revealed a striking differential expression of 1148 genes between patients who were subsequently complete (CR) or non-complete responders (non-CR) to AZA therapy (Figure 1A). Using a Fluidigm nanofluidic system, we have validated the differential expression of a subset of these genes between CR and non-CR patients in two independent cohorts, totalling 67 patients, from the U.K. and Sweden. We have additionally confirmed that our gene signature does not simply segregate patients based on disease severity or poor overall survival, but rather uniquely prognosticates best AZA response. Pathway analyses of the differentially expressed genes indicates that the HSPCs of non-CR patients have decreased cell cycle progression and DNA damage pathways, while concomitantly possessing increased signalling through integrin and mTOR/AKT pathways. Using computational methods, we have determined that the expression of 15 genes (within the 1148 gene set) is sufficient to separate CRs from non-CRs across independent cohorts (Figure 1B). We have also developed a predictive AZA response algorithm that utilises the expression of these genes to identify potential complete and non-complete responders to AZA with high specificity and sensitivity (Figure 1C). Furthermore, we have identified statistically significant correlations between recurrent DNA mutations in MDS and our prognostic gene signature (SF3B1 & TET2 with CR, STAG2 and NUP98 with non-CR, p

Description: Based on the profile of genetic alterations occurring in tumor samples from selected diffuse large B-cell lymphoma (DLBCL) patients, 2 recent whole-exome sequencing studies proposed partially overlapping classification systems. Using clustering techniques applied to targeted sequencing data derived from a large unselected population-based patient cohort with full clinical follow-up (n = 928), we investigated whether molecular subtypes can be robustly identified using methods potentially applicable in routine clinical practice. DNA extracted from DLBCL tumors diagnosed in patients residing in a catchment population of ∼4 million (14 centers) were sequenced with a targeted 293-gene hematological-malignancy panel. Bernoulli mixture-model clustering was applied and the resulting subtypes analyzed in relation to their clinical characteristics and outcomes. Five molecular subtypes were resolved, termed MYD88, BCL2, SOCS1/SGK1, TET2/SGK1, and NOTCH2, along with an unclassified group. The subtypes characterized by genetic alterations of BCL2, NOTCH2, and MYD88 recapitulated recent studies showing good, intermediate, and poor prognosis, respectively. The SOCS1/SGK1 subtype showed biological overlap with primary mediastinal B-cell lymphoma and conferred excellent prognosis. Although not identified as a distinct cluster, NOTCH1 mutation was associated with poor prognosis. The impact of TP53 mutation varied with genomic subtypes, conferring no effect in the NOTCH2 subtype and poor prognosis in the MYD88 subtype. Our findings confirm the existence of molecular subtypes of DLBCL, providing evidence that genomic tests have prognostic significance in non-selected DLBCL patients. The identification of both good and poor risk subtypes in patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) clearly show the clinical value of the approach, confirming the need for a consensus classification.

Description: Key Points The majority of mutations are found in genes that have low or no detectable biological expression. Mutated genes often show differential allelic expression in multiple myeloma patient samples.

Description: An acquired V617F JAK2 mutation occurs in patients with polycythemia vera (PV) or essential thrombocythemia (ET). In a proportion of V617F-positive patients, mitotic recombination produces mutation-homozygous cells that come to predominate with time. However, the prevalence of homozygosity is unclear, as previous reports studied mixed populations of wild-type, V617F-heterozygous, and V617F-homozygous mutant cells. We therefore analyzed 1766 individual hematopoietic colonies from 34 patients with PV or ET in whom granulocyte sequencing demonstrated that the mutant peak did not predominate. V617F-positive erythroid burst-forming units (BFU-Es) were more frequent in patients with PV compared with patients with ET (P = .022) and, strikingly, V617F-homozygous BFU-Es were detected in all 17 patients with PV, but in none of the patients with ET (P 〈 .001). Moreover, mutation-homozygous cells were present in 2 patients with ET after polycythemic transformation. These results demonstrate that V617F-homozygous erythroid progenitors are present in most patients with PV but occur rarely in those with ET.

Description: Multiple factors are likely to contribute to the pathogenesis of thrombosis in Essential thrombocythaemia (ET) including platelet number, activation of platelets and leukocytes; the formation of platelet leucocyte aggregates, and circulating prothrombotic and endothelial factors. The normal functional activity of platelets depends on the presence of platelet glycoprotein complexes, which play an important role in platelet adhesion and aggregation. A number of polymorphisms in platelet glycoproteins have been correlated with thrombotic events in hematologically normal patients. Particular polymorphisms of interest include three within the glycoprotein Ibα gene: −5T/C (affecting a Kozak sequence), 1018C/T (T145M, encoding Human Platelet Antigen − 2), a variable number of tandem repeats (VNTR) in a region encoding the macroglycopeptide and one polymorphism C807T, within the glycoprotein Ia gene. To date whether there is a relationship between these platelet glycoprotein polymorphisms and the clinical features of ET has not been determined. In this study samples obtained from 797 ET patients recruited to the 3 large prospective PT–1 trials were genotyped for the polymorphisms of interest and the results were correlated with clinical events including haemorrhagic and arterial or venous thrombotic events in the year prior to diagnosis and following trial entry (median 30 months). The VNTR data were analysed using the sum of the number of repeats of both alleles and treated as a continuous variable using logistic regression for retrospective and a Cox survival model for prospective analyses; other polymorphisms were analysed using the Chi-squared test for the retrospective clinical events, and log-rank survival analysis for the prospective clinical events. Neither the C807T, −5T/C Kozak nor the T145M polymorphism was associated with clinical events in the ET patient cohort. Interestingly the number of VNTR repeats was inversely associated with (p=0.02) rate of arterial events after trial entry in this patient cohort, but not for events in the year prior to diagnosis or post trial entry venous thrombotic or haemorrhagic events. This analysis suggested that a decreasing sum of VNTR were associated with an increased risk of arterial thrombosis, the degree of increase in risk was 75% per repeat (95% confidence interval 1.0–2.9). After multivariate analysis of this data with correction for age, sex, JAK2V617F and MPLW515L/K status the effect of the number of VNTR repeats remained significant (p=0.048). These results suggest that the C807T, T145M and −5T/C Kozak polymorphisms do not correlate with clinical events in ET. However a reduction in the number of VNTR within the glycoprotein Ibα gene was progressively associated with arterial events post trial entry (p=0.024) and remained significant on multivariate analysis (p=0.048).

Description: BCR-ABL negative myeloproliferative neoplasms (MPNs), such as polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are chronic myeloid malignancies characterized by overproduction of hematopoietic cells. JAK2 mutations are found in most patients with PV, and in only 50-60% of patients with ET and MF. JAK2 mutation testing has greatly simplified MPN diagnosis, but distinguishing JAK2-wildtype ET from reactive thrombocytosis remains a diagnostic challenge. Mutations in signalling pathways (MPL, LNK) and epigenetic regulators (TET2, DNMT3A, IDH1/2, EXH2, ASXL1) have been found in a minority of MPNs. However genome-wide data are lacking and the pathogenesis of MPNs that do not harbor JAK2 or MPLmutations remains obscure. Methods Exome sequencing was performed in 151 MPN patients on matched tumor and constitutional samples. CALR status was assessed in 3412 samples using Sanger sequencing and analysis of exome/genome sequencing data. Presence of CALR mutations in hematopoietic stem and progenitor cells was assessed by flow sorting and sequencing. Phylogenetic trees were established using hematopoietic colonies. Calreticulin cellular localisation was assessed in patient samples and cell lines expressing CALR variants by flow cytometry and immunofluorescence. Results Exome sequencing identified 1498 somatic mutations with a median of 6.5 mutations in PV and ET, and 13 in MF (MF vs ET, P=0.0002; MF vs PV, P=0.008). JAK2V617F was found in all cases of PV (n=48), 56% of ET (35/62), and 69% of MF (27/39), and MPL mutations in 7 ET and MF cases.  Mutations in epigenetic regulators TET2, DNMT3A, ASXL1, EZH2, and IDH1/2 were identified in 22, 12, 12, 4, 3 patients respectively, and components of the splicing machinery (U2AF1, SF3B1 or SRSF2) were mutated in 9 patients.  Mutations in rare genes reported to be mutated in MPNs were found in four patients (1 CBL; 2 NFE2; 1 SH2B3/LNK). We found novel somatic mutations in CHEK2 (1 PV, 1 ET and 1 MF) which have not been previously reported in MPNs.  The mutation spectrum showed a predominance of C〉T transitions. Pairwise associations between MPN genes demonstrated that ASXL1 and SRSF2 mutations were positively correlated with mutations in epigenetic modifiers. Novel somatic mutations in calreticulin (CALR) were identified by exome sequencing in the majority (26/31) of JAK2 or MPL unmutated patients. CALR and JAK2/MPL mutations were mutually exclusive, and 97% of patients harbored a mutation in 1 of these 3 genes. In an extended follow up screen of 1345 hematological malignancies, 1517 other cancers and 550 controls we found CALR mutations in 71% of ET (80/112), 56% of idiopathic MF (18/32), 86% of post ET-MF (12/14) and 8% of myelodysplasia (10/115), but not in other myeloid, lymphoid or solid cancers. Compared to JAK2-mutated MPNs, those with CALR mutations presented with higher platelet counts (Wilcoxon rank-sum, P=0.0003), lower hemoglobin levels (Student’s t test, P=0.02) and showed a higher incidence of transformation to MF (Fishers exact, P=0.03). All CALR mutations were insertions or deletions affecting exon 9, with 2 common variants L367fs*46 (52 bp deletion) and K385fs*47 (5 bp insertion). Loss of heterozygosity over CALR was seen in a minority of patients. Of 148 CALR mutations identified, there were 19 distinct variants. Remarkably, all generated a +1 basepair frameshift, which results in loss of most of the C-terminal acidic domain of the protein as well as the KDEL Golgi-to-endoplasmic reticulum (ER) retrieval signal, raising the possibility of compromised ER retention. Mutant proteins were readily detected in transfected cell lines and localised to the ER in the same manner as wildtype CALR, without Golgi or cell surface accumulation. These results are consistent with studies reporting KDEL-independent mechanisms of ER retention. Mutation of CALR was detected in highly purified hematopoietic stem/progenitor cells. Clonal analyses demonstrated CALR mutations in the earliest phylogenetic node in 5/5 patients, consistent with it being an initiating mutation in these individuals. Conclusions We describe the mutational landscape of BCR-ABL negative MPNs and demonstrate that somatic mutations in the endoplasmic reticulum chaperone CALR are found in the majority of patients with JAK2-unmutated MPNs. These results reveal a novel biological pathway as a target for tumorigenic mutations and will simplify diagnosis of MPN patients. Disclosures: Bowen: Celgene: Honoraria. Harrison:S Bio: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Shire: Speakers Bureau; Celgene: Honoraria; YM Bioscience: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Vannucchi:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Description: Multiple myeloma (MM) is an incurable plasma cell malignancy with a complex and incompletely understood molecular pathogenesis. It is classically subdivided into a subtype with rearrangements involving the IGH locus and the hyperdiploid subtype, which harbors multiple trisomies involving odd-numbered chromosomes. These chromosomal abnormalities are nevertheless insufficient for malignant transformation because they are also observed in monoclonal gammopathy of unknown significance, a pre-malignant syndrome that precedes MM. Malignant progression is associated with additional events such as somatic mutations, aberrant methylation, and chromosomal copy-number changes. This implies that the genetic landscape of MM changes over time, driving its symptomatic emergence and progression. Furthermore, evidence suggests that the molecular events necessary for myeloma progression are not attained in a linear fashion but rather through branching, nonlinear pathways. Through whole exome and genome sequencing, copy number profiling and cytogenetics, it has been possible to identify new candidate genes, define diverse mutational processes contributing to the genomic landscape, and study patterns of disease evolution. In particular, the overwhelming majority of MM patients have at least one cluster of subclonal variants, including subclonal driver mutations, implying ongoing tumor evolution. Serial samples reveal diverse patterns of clonal evolution, including linear evolution, differential clonal response and branching evolution. These new findings reveal the myeloma genome to be heterogeneous across patients and within individual patients and that it can exhibit diversity in clonal admixture and dynamics in response to therapy. As modern genomics technologies will increasingly become available in routine diagnostic laboratories, it will be critical to develop an understanding of myeloma as a restless and dynamic disease, with multiple subclones exploring the genomic landscape seeking fitter states. This understanding should, in turn, be used to shape our therapeutic approach to the disease. Disclosures: No relevant conflicts of interest to declare.

Description: Ring sideroblasts (RS) characterize a group of myelodysplastic syndromes (MDS) categorized in the WHO classification as refractory anemia with RS (RARS) or refractory cytopenia with multilineage dysplasia and RS (RCMD-RS), according to the presence of 15% or more bone marrow RS and dysplasia in one or more myeloid lineages. A high prevalence of somatic mutations in SF3B1 was reported in MDS with RS [N Engl J Med 2011;365:1384-95], and recent unsupervised analyses suggested that MDS with SF3B1 mutation represent a homogeneous subset [Blood 2014 Jun 26]. In this study, we performed a comprehensive mutation analysis of genes implicated in myeloid disorders in a large and well-characterized cohort of myeloid neoplasms with 1% or more RS with the aim to identify mutation patterns that affect disease phenotype and clinical outcome. The study population consisted of 309 patients (pts), including: a) 244 with MDS, of whom 160 assigned to sideroblastic categories (RARS, RCMD-RS) and 84 to other WHO categories [34 RA or RCMD, 7 MDS with isolated del(5q), 20 RAEB-1, 23 RAEB-2]; b) 51 with myelodysplastic/myeloproliferative neoplasms (MDS/MPN: 9 CMML, 42 RARS-T); c) 14 with AML-MDS. SF3B1 mutations were observed in 151/244 pts with MDS and RS (62%). Within sideroblastic categories, SF3B1 mutation was found in 81/91 cases of RARS (89%), and 48/69 RCMD-RS (70%). Among pts classified in other MDS categories, significantly lower rate of SF3B1 mutations (22/84, P

Description: Deregulated expression of the type I cytokine receptor, cytokine receptor-like factor 2 (CRLF2 -d), is observed in 5% of B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). It occurs via three known aberrations; a cryptic reciprocal translocation with the immunoglobulin heavy chain locus (IGH); an interstitial deletion within PAR1, resulting in the P2RY8-CRLF2 fusion; rare but recurrent CRLF2 mutation (F323C). All three result in overexpression of CRLF2, however alone it is insufficient to cause overt leukaemia. Mutations of the Janus kinase (JAK) family genes and the IL7 receptor, are recurrent (50% of CRLF2-d) and together with CRLF2 -d result in transformation of mouse BaF3 cells. We noted that outcome data from different study groups were inconsistent, with patients being classified as either high or intermediate risk. Thus, in this largest study to date, our aim was to acertain whether differences in clinical and/or genetic features between patients with CRLF2-d and involvement of either IGH or P2RY8 may define them as different disease entities and partly explain the outcome heterogeneity. Among 160 CRLF2-d patients, chromosomal analysis confirmed a high incidence of additional somatic copies of chromosomes X (39/88, 44%) and 21 (23/88, 26%) and identified that aneuploidy of chromosomes 9 (5/88, 6%) and 17 (7/88, 8%) were recurrent in both groups. From comparison of patients with IGH-CRLF2 and P2RY8-CRLF2, we noted a higher frequency of IKZF1 (79% v 37% p